Antibiotic A16884

ABSTRACT

Antibiotic A16884 and its salts, having antibacterial and anthelmintic activity, prepared by fermentation of Streptomyces lipmanii NRRL 3584.

CROSS-REFERENCE TO RELATED APPLICATION

This is a division of our copending application Ser. No. 60,556, filedAug. 3, 1970, and issued Mar. 6, 1973, as U.S. Pat. No. 3,719,563, whichwas in turn a continuation-in-part of our then co-pending applicationSer. No. 847,923, filed Aug. 6, 1969, and abandoned after the filing ofsaid application Ser. No. 60,556.

SUMMARY OF THE INVENTION

Antibiotic A16884 is a new antibiotic produced by the fermentation of anantibiotic A16884-producing strain of Streptomyces lipmanii. The saltsof A16884 are readily obtained by reaction of A16884 with a suitableacid or base. Antiobiotic A16884 and its salts exhibit antibacterial andanthelmintic activity. The antibacterial activity is exhibited againstboth gram-negative and gram-positive organisms, as well as againstplant-pathogenic organisms.

DESCRIPTION OF PREFERRED EMBODIMENTS

Antibiotic A16884, a sulfur-containing peptide antibiotic, is anamphoteric molecule produced by cultivating under controlled conditionsa hitherto undescribed strain of Streptomyces lipmanii NRRL 3584.

As is the case with many antibiotic-producing cultures, fermentation ofan antibiotic A16884-producing strain of Streptomyces lipmanii resultsin the production of a number of antibiotic substances. AntibioticA16884 is one of the substances. Other substances are either relativelyunstable or are present in only very minor quantities.

Antibiotic A16884 can be utilized as such or as a salt, for example, anacid addition salt or a salt with a cation. In the instance of a saltwith a cation, the salt can be either a mono or di salt. It is oftenpreferred to prepare salts directly in the purification process so thatthe antibiotic as separated is in salt form. Antibiotic A16884 has beenseparated in this manner, and for that reason, is hereinbelowcharacterized as the monoammonium salt.

The monoammonium salt of antibiotic A16884 is a white, amorphous solid,decomposing at about 180°C., very soluble in water, soluble indimethylsulfoxide (DMSO), slightly soluble in lower alkanols, andessentially insoluble in acetonitrile and other organic solvents. Thespecific optical rotation [α]_(D) ²⁵ of the monoammonium salt ofantibiotic A16884, dried at room temperature in vacuo over anhydrouscalcium chloride for about 15 hours, was found to be +140.9° (C = 1percent, w./v. in water).

Electrometric titration of the monoammonium salt of antibiotic A16884 ina 66 percent dimethyl formamide-water solution at an initial pH of 6.6revealed the presence of four titratable groups: pK'a₁ = 3.5; pK'a₂ =5.2; and pK'a₃ = 9.2; and pK'a₄ = 10.3. On like titration of a latersample, except at an initial pH of 5.8, the respective values were pK'a₁= 3.9; pK'a₂ = 5.3; pK'a₃ = 9.2; and pK'a₄ = 10.5. When the monoammoniumsalt of antibiotic A16884 is converted to the acid form, the pK'a at 9.2disappears. The molecular weight of the monoammonium salt calculatedfrom the titration data is about 435.

Elemental analysis of the monoammonium salt of A16884, dried in vacuo atabout 80°C. over phosphorus pentoxide, gave the following values:

    Element          Percent                                                      ______________________________________                                        Carbon           44.01                                                        Hydrogen         5.73                                                         Nitrogen         10.65                                                        Oxygen           31.27                                                        Sulfur           6.86                                                         ______________________________________                                    

Analysis shows a methoxyl content of 6.64 percent, and an acetyl contentof 9.23 percent; and a Van Slyke test for amino nitrogen shows 5.09percent.

The infrared absorption spectrum of the monoammonium salt of antibioticA16884 in a mineral oil mull is shown in FIG. 1 of the accompanyingdrawing. The distinguishable bands in the infrared spectrum over therange of 2.0 to 15.0 microns are as follows: 3.18 (broad band), 5.66,6.26, 6.57, 6.89, 7.15, 7.28, 7.40, 7.73, 8.00, 8.14, 8.79, 9.24, 9.65,9.79, and 10.4 microns.

The ultraviolet absorption spectrum of the monoammonium salt ofantbiotic A16884 in aqueous solution shows absorption maxima at 242 (E₁cm .sup. 1 % = 126) and at 265 mμ (E₁ cm .sup. 1 % = 158); circulardichroism was also measured in aqueous solution and showed a positiveCotton effect at 263 mμ and a negative Cotton effect at 236 mμ.

Paper chromatography of the monoammonium salt of antibiotic A16884 onWhatman No. 1 paper gave an R_(f) value of 0.79 in a solvent system ofpropanol, acetonitrile, and water in a volume ratio of 1:1:1.Bioautographs were obtained by placing the paper chromatograph on agarplates seeded with sensitive organisms, such as Salmonella gallinarum,as test organisms.

The NMR spectrum of A16884 in D₂ O showed the following characteristics:5.16 ppm. (1H, singlet); 4.86; 4.68 ppm. (2H, AB quartet, J = 12.5 Hz);3.9-3.7 ppm. (1H, multiplet); 3.67,3.29 ppm. (2H, AB quartet, J = 18Hz); 3.53 ppm. (3H, singlet); 2.6-2.3 (2H, multiplet); 2.10 ppm. (3H,singlet); 2.1-1.6 ppm. (4H, multiplet).

Paper chromatography of the monoammonium salt was also carried out inother solvent systems with the following results:

      Solvent System          R.sub.f value                                       ______________________________________                                        Ethanol:water (80:20) with 1.5% sodium                                        chloride, paper impregnated with 1N                                           sodium sulfate            .58                                                 Methanol:propanol:water (6:2:1), paper                                        buffered with 0.75 M potassium phos-                                          phate, pH 4.0             .21                                                 Propanol:pyridine:acetic acid:acetoni-                                        trile:water (45:30:9:40:36)                                                                             .40                                                 tert-Amyl alcohol:acetone:water (2:1:2)                                                                 .40                                                 Ethyl acetate:acetic acid:water (3:1:1)                                                                 .36                                                 Methyl ethyl ketone:water (92:8), paper                                       buffered with 0.1 N sodium acetate,                                           pH 4.6                    immobile                                            Propanol:water (70:30)    .30                                                 Butanol saturated with water                                                                            immobile                                            Butanol saturated with water plus 2%                                          p-toluenesulfonic acid    .60                                                 ______________________________________                                    

When the monoammonium salt of A16884 is subjected to thin-layerchromatography on silica gel plates in 70 percent aqueous acetonitrile,utilizing a ninhydrin spray as a detector, it has an R_(f) value ofabout 0.47; on cellulose plates in 70 percent aqueous acetonitrile,utilizing the same procedure for detection, it has an R_(f) value of0.45.

Amino acid analysis of an acid hydrolysate of antibiotic A16884, run bythe Spackman-Moore-Stein technique, showed two ninhydrin reacting peaks,one of which was eluted identically with glycine (0.758 μmoles/mg.), theother of which was eluted just prior to glycine and was identified asα-aminoadipic acid (2.39 μmoles/mg.). On like analysis of a latersample, the values observed were 0.49 μmoles/mg. and 1.2 μmoles/mg.,respectively.

A number of qualitative chemical tests have been carried out with theantibiotic A16884. Antibiotic A16884 gives a positive test withninhydrin, Pan Dutscher, Benedict, Molisch, iodine and dansyl chloridereagents, but not with Fehling, ferric chloride, biuret, and Sakaguchireagents.

The monoammonium salt of antibiotic A16884 is stable at pH 3-9 at 5°C.for 8 days; relatively stable at pH 3-9 at 25°C. for 4 days; andunstable at varying pH values at 100°C. within 5 minutes. Biologicalactivity is slowly lost at pH 3-9 at a temperature of 37°C., half beinglost at 4 days.

Based on the various foregoing physical characteristics the structure ofantibiotic A16884 has been determined to be as follows: ##SPC1##

which structure is designated7-(5-amino-5-carboxyvaleramido)-7-methoxycephalosporanic acid.

Antibiotic A16884 has an inhibitory action against the growth of bothgram-positive and gram-negative bacteria. The levels at which partiallypurified monoammonium salt of antibiotic A16884 shows inhibition againstthe growth of illustrative organisms are set forth in Table I. Theinhibitory levels were determined by the agar-dilution test or by thebroth-dilution test (identified in the table by the letters "ad" and"bd", respectively).

In the agar-dilution test, the test organism was streaked on a series ofagar plates containing various concentrations of the monoammonium saltof antibiotic A16884 to determine the minimum concentration in mcg./ml.(micrograms per milliliter) in the agar substrate which inhibited thegrowth of the organism over a period of forty-eight hours (seventy-twohours in the case of the plant pathogen organisms).

In the broth-dilution test, a series of tubes containing nutrient brothcontaining varied concentrations of the ammonium salt of antibioticA16884 were inoculated with the test organism to determine the minimumconcentration of the monoammonium salt of A16884 in mcg./ml in the brothsubstrate which inhibited organism growth for a period of about twentyhours.

                  TABLE I                                                         ______________________________________                                                           Inhibitory Concentration                                   Test Organism      mcg./ml.                                                   ______________________________________                                        Escherichia coli EC 0127                                                                         6.25 a.d.                                                  Proteus PR6        1.56 a.d.                                                  Proteus PR4        3.12 a.d.                                                  Salmonella typhimurium 54                                                                        3.12 a.d.                                                  Salmonella typhosa T63                                                                           1.56 a.d.                                                  Staphylococcus aureus 3055                                                                       50.00 a.d.                                                 Staphylococcus aureus 3150                                                                       >50.00 a.d.                                                Pseudomonas aeruginosa X239                                                                      >50.00 a.d.                                                Salmonella flexneri SH3                                                                          6.25 a.d.                                                  Klebsiella aerobacter K1                                                                         6.25 a.d.                                                  Klebsiella aerobacter KA14                                                                       1.56 a.d.                                                  Mycobacterium avium X85                                                                          >50.00 a.d.                                                Streptococcus pyogenes C203                                                                      3.12 a.d.                                                  Bacillus subtilis X12.1                                                                          3.12 a.d.                                                  Neurospora sp. M45-846                                                                           >50.00 a.d.                                                Sarcina lutea X186 6.25 a.d.                                                  Escherichia coli EC0127                                                                          7.80 b.d.                                                  Klebsiella aerobacter KA14                                                                       15.60 b.d.                                                 Salmonella typhosa SA12                                                                          31.20 b.d.                                                 ______________________________________                                         No binding by horse serum was noted in any of the above tests.           

As can be seen from the above table, antibiotic A16884 as themonoammonium salt exhibits activity against gram-positive andgram-negative bacterial organisms.

More highly purified monoammonium salt of antibiotic A16884 was furtherevaluated for antibacterial activity in a test employing the brothdilution technique described above. The results, expressed in terms ofthe minimum number of micrograms per milliliter required to obtaininhibition, were as set forth below in Table II.

                  TABLE II                                                        ______________________________________                                                           12-hour    24-hour                                         Organism           reading    reading                                         ______________________________________                                        Streptococcus pyogenes C203                                                                       64        128                                             Staphylococcus aureus 3055                                                                       128        >128                                            Escherichia coli EC14                                                                             8          8                                              Klebsiella aerobacter sp. KA14                                                                    8          16                                             Proteus sp. PR6    ND*         8                                              Proteus sp. PR17    2          8                                              Salmonella typhosa SA12                                                                          ND*         4                                              Salmonella typhimurium S4                                                                        ND*         4                                              Pasteurella multocida P3                                                                         ND*         2                                              Shigella sonnia I SH10                                                                           ND*         16                                             ______________________________________                                         *ND -- Not done.                                                         

Antibiotic A16884 and its salts also exhibit in vivo activity against anumber of the above organisms and hence are useful in controllinginfections caused by such organisms in host animals. Partially purifiedantibiotic A16884 as the monoammonium salt exhibited an ED₅₀ of 23mg./kg. in mice infected with Proteus PR6, and an ED₅₀ of 33.8 mg./kg.in mice infected with Shigella SH3; more highly purified A16884 as themonoammonium salt exhibited an ED₅₀ of 3.64 mg./kg. in mice infectedwith Escherichia coli EC14, an ED₅₀ of 23 mg./kg. in mice infected withSalmonella typhosa SA12, and an ED₅₀ of 93.4 mg./kg. in mice infectedwith Klebsiella pneumoniae K1. Administration was by the sub-cutaneousroute.

As noted hereinabove, antibiotic A16884 and its salts exhibitanthelmintic activity in addition to antibacterial activity. Henceantibiotic A16884 or the salt thereof can be administered towarm-blooded animals to control various internal parasites, particularlystomach and intestinal worms such as Ascaris lumbricoides var. suum,Nematospiroides dubius, Aspiculuris tetraptera, Syphacia obvelata, andthe like. The administration is preferably by the oral route, forexample, by inclusion of antibiotic A16884 or a salt in animal feed, byadministration of tablets, drenches, etc. containing A16884 or a salt,or by other means. In general, doses of from 1 to 500 milligrams perkilogram or more of animal body weight are effective in single doseadministration. Where antibiotic A16884 or a salt thereof is supplied asa constituent of a regular feed, concentrations of from 0.0001 to 0.05percent or more give good results. A preferred range of concentration ofantibiotic A16884 or a salt thereof in feeds is from 0.01 to 0.05percent.

The anthelmintic activity of antibiotic A16884 is illustrated by thefollowing evaluations.

In a first evaluation, antibiotic A16884 monoammonium salt wasadministered in a single dose by gavage to each of two mice infectedwith Aspiculuris tetraptera and Syphacia obvelata (pinworms). The dosewas 500 milligrams of antibiotic A16884 monoammonium salt per kilogramof individual animal body weight, administered in a suspension ofphysiological saline containing 0.125 percent of methylcellulose assuspending agent. A control group of mice infected with Aspiculuristetraptera and Syphacia obvelata was employed in the evaluation. Bothgroups were maintained under normal laboratory conditions for 48 hours,following the dosing of the treated group. All mice were then sacrificedand examined to determine the presence and numbers of pinworms, whichwere as reported in the following table:

                  TABLE III                                                       ______________________________________                                                         Number of                                                                     Pinworms Per Animal                                                           Aspiculuris                                                                             Syphacia                                                            tetraptera                                                                              obvelata                                           ______________________________________                                        Control            26          52                                             Antibiotic A16884 monoammonium                                                salt at 500 mg./kg.                                                                               0          2.5                                            ______________________________________                                    

In another evaluation, antibiotic A16884 monoammonium salt was mixedwith standard mouse feed to obtain a plurality of treated feeds,containing antibiotic A16884 monoammonium salt in concentrations of0.005, 0.01, and 0.05 percent by weight. The feeds were utilized asdiets for separate groups of mice, five mice per group. About 24 hoursafter initiation of the feeding, the mice were infected with Ascarislumbricoides var. suum ova. Another group of five mice was fed thenon-medicated feed to serve as a control but was similarly infected atthe time with Ascaris lumbricoides var. suum. All groups were fed theirrespective feed and maintained under normal laboratory conditions for aperiod of ten days, at which time, all mice were taken off feed. On theeleventh day, all mice were sacrificed and the lungs examined todetermine the presence and, if present, numbers of lesions of Ascarislumbricoides var. suum.

The level of antibiotic A16884 monoammonium salt in the diet and theaverage number of lung lesions per animal in each group are set forth inthe following table:

                  TABLE IV                                                        ______________________________________                                                           Average Number of Lung                                     Group              Lesions Per Group                                          ______________________________________                                        Control            2.2                                                        Antibiotic A16884 monoammonium                                                salt at 0.005 percent                                                                            0.5                                                        Antibiotic A16884 monoammonium                                                salt at 0.01 percent                                                                             0.4                                                        Antibiotic A16884 monoammonium                                                salt at 0.05 percent                                                                             0.4                                                        ______________________________________                                    

Antibiotic A16884 can be produced by culturing a newly found andhitherto undescribed organism strain isolated from soil samples obtainedfrom South America.

The organism was isolated from the above soil samples by suspendingportions of the soil samples in sterile distilled water, and bystreaking the suspensions on nutrient agar. The seeded nutrient agarplates were incubated at about 25°-35°C. for several days. At the end ofthe incubation period, colonies of the antibiotic A16884-producingorganism were transferred with a sterile platinum loop to agar slants.The agar slants were then incubated to provide suitable amounts ofinoculum for the production of antibiotic A16884.

The actinomycete used according to this invention for the production ofantibiotic A16884 has been designated as a strain of Streptomyceslipmanii Waksman and Curtis.

The novel organism capable of producing antibiotic A16884 has beenplaced on permanent deposit without restriction as to availability withthe culture collection of the Northern Utilization Research andDevelopment Division, Agricultural Research Service, U.S. Department ofAgriculture (Formerly Northern Regional Research Laboratories), Peoria,Illinois 61604, and is available to the public under culture No. NRRL3584.

The characteristics of Streptomyces lipmanii NRRL 3584 are given in thefollowing tables. The methods recommended for the InternationalStreptomyces Project (Shirling et al, "Methods for Characterization ofStreptomyces Species," Intern. Bull. Systematic Bacteriol. 16: 313-340[1966]) for the characterization of Streptomyces species have been usedalong with certain supplementary tests. Color names were assignedaccording to the ISCC-NBS method described by Kelly et al. in TheISCC-NBS Method of Designating Colors and a Dictionary of Color Names(U.S. Department of Commerce Circ. 553, Washington, D.C. 1955). Figuresin parenthesis refer to the Tresner and Backus color series (Tresner etal., "System of Color Wheels for Streptomyces Taxonomy," Appl.Microbiol. 11: 335-338 [1963]) and color tab designations areunderlined. The Maerz and Paul color blocks (Maerz et al., Dictionary ofColor (McGraw-Hill Book Co., Inc., New York, 1950) are enclosed inbracket. Cultures were grown at 30°C. for 14 days unless notedotherwise.

                  TABLE V                                                         ______________________________________                                        Property Observed                                                                            Characteristics of A16884                                      ______________________________________                                        Morphology     Sporophores are usually straight                                              to flexuous with occasional hooks                                             produced; spores are short, cylin-                                            drical, 0.5-1.5μ × 1.0-2.5μ, and                                  occur usually in chains of 3-10                                               and occasionally from 10-50.                                                  Spores are smooth in outline as                                               observed by electron microscopy.                               Culture                                                                       Characteristics on:                                                           ISP No. 2 (Yeast-Malt                                                                        Growth moderate, reverse dark                                  Extract Agar)  grayish brown [8H9]; aerial my-                                               celium pale yellow (Y) 2db                                     ISP No. 3 (Oatmeal                                                                           Growth moderate, reverse dark                                  Agar)          grayish yellow [13E4]; aerial                                                 mycelium moderate, white (W)                                                  13ba to pale yellow (Y) 2db.                                   ISP No. 4 (Inorganic                                                                         Growth moderate, reverse brownish                              salts and Soluble                                                                            gray [7C7]; aerial mycelium mod-                               Starch Agar)   erate, pale yellow (Y) 2db.                                    ISP No. 5 (Glycerol-                                                                         Growth abundant, reverse light                                 Asparagine Agar)                                                                             yellowish brown [13I7]; aerial                                                mycelium abundant, grayish yellow                                             ish pink (R) 5dc.                                              Tomato paste-oatmeal                                                                         Growth abundant, reverse grayish                               Agar           yellowish brown [15E8]; aerial                                                mycelium abundant, yellowish                                                  gray (GY) 2dc.                                                 Emersons' Agar Growth moderate, reverse dark                                                 grayish yellowish brown [8E9];                                                aerial mycelium and spores absent                              Bennetts' Agar Growth abundant, reverse medium                                               yellowish brown [14E7]; aerial                                                mycelium abundant, grayish yellow                                             (R) 3ec.                                                       Czapeks' Agar  Growth scant, white; scant aerial                                             mycelium (W) 13ba.                                             Glucose-asparagine                                                                           Growth abundant, reverse grayish                               Agar           yellow [12D4]; aerial mycelium                                                abundant, yellowish gray (GY)                                                 2dc.                                                           Tyrosine Agar  Growth moderate, reverse light                                                yellowish brown [12C5]; aerial                                                mycelium abundant, grayish yellow                                             (R) 3ec.                                                       Nutrient Agar  Moderate growth, reverse pale                                                 yellow [11C1]; no aerial mycelium.                             Calcium Malate Agar                                                                          Moderate growth, reverse black                                                [56C1]; very scant aerial mycel-                                              ium.                                                           Physiology                                                                    Action on milk Coagulation, peptonization.                                    Nitrate reduction                                                                            Positive                                                       Melanin production                                                             Peptone-iron agar                                                                           Negative                                                        Tryptone-yeast ext.                                                                         Negative                                                       broth                                                                         Temperature requirements                                                                     Abundant growth and sporulation                                on tomato paste-oatmeal                                                                      at 26°C. and 30°C; slight growth                 agar           at 37°C; no growth at 43°C.                      Response of vegetative                                                        color to pH change                                                             0.05N HCl     Brownish-gray pigment changes                                                 to red                                                          0.05N NaOH    No change.                                                     Gelatin liquefaction                                                                         100%                                                           ______________________________________                                    

In Table VI are set forth the results of carbon utilization testscarried out on organism NRRL 3584. In the table, the following symbolsare employed:

+ = growth and utilization

- = no growth, no utilization

                  TABLE VI                                                        ______________________________________                                        Carbon Utilization Pattern for NRRL 3584                                      Compound          Growth Response                                             ______________________________________                                        L-arabinose       -                                                           sucrose           -                                                           D-xylose          +                                                           D-fructose        -                                                           glucose           +                                                           rhamnose          -                                                           raffinose         -                                                           i-inositol        -                                                           D-mannitol        -                                                           Control (no carbon)                                                                             -                                                           ______________________________________                                    

As noted above, antibiotic A16884 can be produced by the cultivation ofNRRL 3584. The culture medium employed in producing antibiotic A16884 bycultivation of the above-identified organism can be any of severalmedia, since, as is apparent from the above-described utilization tests,the organism is capable of utilizing different energy sources. However,for economy of production, maximum yield of antibiotic, and ease ofisolation of the antibiotic, certain relatively simple nutrient sourcesare preferable. For example, the media which are useful in theproduction of the antibiotic include an assimilable source of carbonsuch as glucose, starch, glycerine, molasses, dextrin, and the like. Thepreferred source of carbon is glucose. Additionally, employable mediainclude a source of assimilable nitrogen such as soybean meal, cornsteep solids, yeast, cottonseed meal, beef extract, peptones (meat orsoy), casein, amino acid mixtures, and the like. Preferred sources ofnitrogen are peptones, soybean meal, amino acid mixtures, and the like.Among the nutrient inorganic salts which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, ammonium, calcium, phosphate, sulfate, chloride, carbonate,and like ions.

Minor elements necessary for optimum growth and development of theorganism used for the production of antibiotic A16884 can also beincluded in the culture medium. Such trace elements commonly occur asimpurities in the other constituents of the medium in amounts sufficientto meet the growth requirements of the actinomycete employed in thisinvention.

The initial pH of the culture medium can be varied. However, it has beenfound desirable that the initial pH of the medium be between 6.5 and7.2. As has been observed with other actinomycetes, the pH of the mediumgradually increases throughout the growth period of the organism whilethe antibiotic is being produced, and may attain a level of from 6.7 to7.5 or above, the final pH being dependent at least in part on theinitial pH of the medium, the buffers present in the medium, and theperiod of time the organism is permitted to grow.

Submerged, aerobic cultural conditions are the conditions of choice forthe production of antibiotic A16884. For preparation of relatively smallamounts, shake flask and surface culture in bottles can be employed; butfor the preparation of large amounts, submerged aerobic culture insterile tanks is preferred. The medium in the sterile tank can beinoculated with a sporulated suspension, but because of the growth lagexperienced when a sporulated suspension is used as the inoculum, thevegetative form of the culture is preferred. By thus avoiding the growthlag, more efficient use of the fermentation equipment is realized.Accordingly, it is desirable first to produce a vegetative inoculum ofthe organism by inoculating a relatively small quantity of culturemedium with the spore form of the organism; and when a young, activevegetative inoculum has been obtained, to transfer the vegetativeinoculum aseptically to the large tank. The medium in which thevegetative inoculum is produced can be either the same as or differentfrom the medium utilized for the large-scale production of antibioticA16884.

The organism which produces antibiotic A16884 will grow over a widetemperature range between 25°-37°C. Optimal production of A16884 seemsto occur at temperatures of 26°-30°C. In general, maximum production ofthe antibiotic occurs within about 36- 72 hours after inoculation of theculture medium.

As is customary in aerobic, submerged culture processes, sterile air isblown through the culture medium. For efficient growth of the organismand antibiotic A16884 production, the volume of air employed in the tankproduction of A16884 is from 0.2 to 0.4 volume of air per minute volumeof culture. The preferred volume is 0.40 volume of air per minute pervolume of culture medium.

The concentration of antibiotic activity in the culture medium can befollowed readily during the fermentation period by testing samples ofthe culture medium for their inhibitory activity against the growth oforganisms known to be inhibited by the presence of antibiotic A16884.The organisms Sarcina lutea and Salmonella galinarum have been found tobe useful for this purpose. The testing of the samples can be carriedout by the well-known turbido-metric or disco-plate methods.

In general, maximum production of A16884 occurs within one to three daysafter inoculation of the culture medium in submerged aerobic culture orshake flask culture processes.

The antibiotic activity produced during the fermentation of A16884occurs in the antibiotic broth. Accordingly, isolation techniquesemployed in the production of A16884 are designed to permit maximumrecovery of the antibiotic from the broth. Thus, for example, myceliumand undissolved solids are removed from the fermentation broth byconventional means such as filtration or centrifugation, and antibioticA16884 can be recovered from the filtered or centrifuged broth byemploying extraction or adsorption technique.

For the recovery of A16884 by adsorption techniques, various adsorbentsand ion exchange resins can be used, for example, carbon, silica gel,alumina, and ion exchange resins. Antibiotic A16884 as obtained fromfermentation may be either amphoteric or salt form, depending uponfermentation conditions. Regardless of which form, it can be adsorbedonto one of the above or similar adsorbents from solution in a suitablesolvent. The adsorbed antibiotic A16884 or salt can then be eluted fromthe adsorbent by suitable elution techniques, such as by washing theadsorbent on which the antibiotic A16884 or salt thereof is adsorbedwith a solvent. Where the elution is carried out by washing with asolution of, e.g., ammonium formate or sodium acetate, the processresults in elution of antibiotic A16884 as the ammonium or sodium salt,respectively. Such salts are readily converted back to antibiotic A16884in conventional procedures. In the foregoing recovery procedure,microcrystalline cellulose can also be used as adsorbent.

Salts of antibiotic A16884 other than ammonium or alkali metal arepreferably prepared by conventional reaction of antibiotic A16884 inunmodified amphoteric form with the respective acid or base. Thus, inpreparing acid addition salts, antibiotic A16884 is reacted with aninorganic or organic acid. Representative suitable acids includehydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid,phosphoric acid, acetic acid, benzoic acid, sulfamic acid, tartaricacid, citric acid, maleic acid, succinic acid, ascorbic acid, andglycolic acid.

Antibiotic A16884 also forms salts with cations by reaction of A16884 inunmodified, amphoteric form with inorganic and organic bases and salts.Exemplary of these salts are ammonium and substituted ammonium salts;alkali metal salts, such as sodium potassium, lithium, cesium, andrubidium; alkaline earth metal salts such as calcium, strontium, andbarium; and salts with other metals such as aluminum, copper, zinc,magnesium, and silver. In respect to organic bases, the identity of thebase is not critical, although, in general, a base having a pH of,numerically 3.0 or above in water is preferred. Representative suitableorganic bases include benzylamine, methylamine, diethylamine,triethylamine, procaine, diisopropylamine, ethanolamine,cyclohexylamine, dicyclohexylamine diphenylamine, di-n-butylamine,quinoline, and pyridylamine.

The salts of antibiotic A16884 which are pharmaceutically acceptable aregenerally preferred. However, all salts are useful as intermediates inthe production, separation, and purification of antibiotic A16884. Fortherapeutic purposes, either cationic or anionic pharmaceuticallyacceptable salts are generally equivalent to antibiotic A16884; however,particular salts are occasionally preferred due to a favorable property,such as solubility, conferred by the salt-forming moiety.

In order to illustrate more fully the operation of the invention, thefollowing examples are provided by way of illustration.

EXAMPLE 1 SHAKE FLASK PRODUCTION OF ANTIBIOTIC A16884

A sporulated culture of Streptomyces lipmanii NRRL 3584 was produced bygrowing the organism on a nutrient agar slant having the followingcomposition:

    Dextrin                 10.00 g.                                              Yeast Extract            1.00 g.                                              Hydrolyzed Casein                                                             ("N-Z Amine-Type A," Sheffield                                                Chemical Company)        2.00 g.                                              Beef Extract             1.00 g.                                              Meer Agar (washed three times)                                                                        20.00 g.                                              Deionized water          1 liter                                          

The pH of the medium was adjusted to pH 7.0 by the addition of sodiumhydroxide.

The agar slant was inoculated with spores of Streptomyces lipmanii NRRL3584 and was incubated for 6 days at 30°C. The agar slant was thencovered with sterile distilled water and gently scraped to remove thespores and cells as an aqueous suspension thereof. One milliliter of theresulting suspension was used to inoculate each 100 ml. portion of avegetative medium having the following composition:

    Glucose                 15.00 g.                                              Soybean meal            15.00 g.                                              Cornsteep solids         5.00 g.                                              Calcium carbonate        2.00 g.                                              Sodium chloride          5.00 g.                                              Deionized water          1 liter                                          

The pH of the vegetative medium was adjusted to pH 6.7 by the additionof sodium hydroxide.

The vegetative inoculum was shaken for 36 hours at 30°C. on a reciprocalshaker with a two-inch stroke at 108 rpm. The inoculum so prepared wasthen utilized in the production of A16884 as follows.

A production medium was prepared having the following composition:

    Soybean meal            15.00 g.                                              Casein                   1.00 g.                                              Sodium nitrate           3.00 g.                                              Glucose syrup                                                                 (50 percent glucose)    20.00 g.                                              Tap water                1 liter                                          

One hundred milliliter portions of the production medium were placed in500 milliliter Erlenmeyer flasks which were sterilized at 120°C. for 30minutes. When cooled, each flask was inoculated with a five percentvegetative inoculum. The fermentation was shaken for 72 hours at 30°C.on a rotary shaker operating at 250 rpm. During the fermentation, themedium was aerated with sterile air at a rate of 0.4 v./v./min.Isolation was carried out essentially as reported hereinbelow in Example8.

EXAMPLE 2

Antibiotic A16884 was produced according to the process of Example 1,but utilizing a production medium having the following composition:

    Distillers' solubles (Nadrisol)                                                                        5.00 g.                                              Soybean flour (Nutrisoy 200D)                                                                          5.00 g.                                              Peanut meal              5.00 g.                                              Blackstrap molasses      5.00 g.                                              Oatmeal                  5.00 g.                                              Glycerol                10.00 g.                                              Tap water                1 liter                                          

and utilizing instead of a rotary shaker a reciprocal shaker operatingat 108 strokes per minute.

EXAMPLE 3

Antibiotic A16884 was produced according to the process of Example 1,but utilizing a production medium having the following composition:

    Oatmeal              20.00 g.                                                 Glycerol             10.00 g.                                                 Tap water             1 liter                                             

EXAMPLE 4

Antibiotic A16884 was produced according to the process of Example 1,but utilizing a production medium having the following composition:

    Cottonseed flour      20.00 g.                                                Glycerol              10.00 g.                                                Glucose                5.00 g.                                                Tap water              1 liter                                            

EXAMPLE 5

Antibiotic A16884 was produced according to the process of Example 1 bututilizing a production medium having the following composition:

    Glucose                 20.00 g.                                              Soluble starch          10.00 g.                                              Peptone (Wilson's 159)  30.00 g.                                              Hydrolyzed casein                                                             ("N-Z amine-type A,"                                                          Sheffield Chemical                                                            Co.)                     4.00 g.                                              Magnesium sulfate heptahydrate                                                                         5.00 g.                                              Sodium carbonate         2.00 g.                                              Tap water               1100 ml.                                          

EXAMPLE 6

Another sporulated culture of Streptomyces lipmanii NRRL 3584 wasproduced by growing the organism on a nutrient agar slant. The slant inthis instance had the following composition:

    Dextrin               10.00 g.                                                Cottonseed flour      10.00 g.                                                Yeast extract          1.00 g.                                                Meer agar             25.00 g.                                                Deionized water       1000 ml.                                            

The pH of the medium was adjusted, by addition of sodium hydroxide, to7.0.

The agar slant was inoculated with spores of Streptomyces lipmanii NRRL3584 and incubated for 7 days at 30°C. The agar slants were then scrapedto remove spores to which were added 2.0 ml. of sterile beef serum. To asterile lyophile tube was then transferred 0.1 ml. of the resultingserum spore suspension; it was freeze-dried in the form of pellets.

The freeze-dried pellets thus obtained were used to inoculate avegetative medium having the following composition:

    Glucose                  5.00 g.                                              Dextrin                 10.00 g.                                              Bacto-tryptone           5.00 g.                                              Yeast extract            5.00 g.                                              Magnesium sulfate heptahy-                                                    drate                    2.00 g.                                              Deionized water          1 liter                                          

The pH of the medium was 6.7 and was left unadjusted.

EXAMPLE 7 PILOT PLANT PRODUCTION OF ANTIBIOTIC A16884

To a 40-liter stainless steel fermentor were added 24 liters of a mediumhaving the following composition:

    Antifoam A (an anti-                                                          foaming agent sold                                                            by Dow Corning)        0.20 g.                                                Glucose                5.00 g.                                                Dextrin 700           50.00 g.                                                Soybean grits         25.00 g.                                                Molasses, blackstrap   3.00 g.                                                Potassium biphosphate  0.25 g.                                                Calcium carbonate      2.50 g.                                                Cold tap water        to 25 liters                                            The initial pH was 6.5 and was not adjusted. The medium was sterilized for     30 minutes at 120°C., cooled, and then inoculated with a five     percent vegetative inoculum produced as in Example 6. The fermentation was     carried out at 30°C. for 66 hours, aerated with sterile air at the     rate of 0.35 v./v./min., and agitated by a mechanical stirrer operated at     420 revolutions per minute. The terminal pH was 7.5.

A16884 was recovered from the broth following the isolation procedureset forth in Example 8.

EXAMPLE 8 ISOLATION OF CRUDE ANTIBIOTIC A16884 AS THE MONOAMMONIUM SALT

Approximately 60 liters of broth obtained as reported in Example 7 wasfiltered with the aid of Hyflo Super-cel (a diatomaceous earth sold byJohns-Manville Products Corporation). The broth filtrate was passed overa 9.6 × 150 cm. column packed with carbon (Pittsburgh Cal. 12 × 40, soldby Pittsburgh Activated Carbon Co.). The column was washed with wateruntil the effluent was colorless, and the activity adsorbed on thecarbon was removed by passing 50 percent aqueous acetone over thecolumn. The fractions containing the activity were combined,concentrated in vacuo to remove acetone, and applied to a 5.9 × 104 cm.column packed with IRA-68 resin (formate cycle) (an anion exchange resinsold by Rohm and Haas Co. and subsequently washed with formic acid toconvert the resin to the formate cycle). The column was washed withwater until the effluent was clear and colorless, and the activity wasremoved by washing with 0.1M ammonium formate solution. The activefractions were combined, and passed over a 4.3 × 72 cm. carbon(Pittsburgh 12 × 40) column. The column was washed with six columnvolumes of water, and the activity was eluted with 30 percent aqueousacetonitrile. The active fractions were combined, concentrated in vacuoto remove acetonitrile, and freeze dried. The yield was 25-30 grams ofsolids.

The freeze-dried preparation was dissolved in a minimum of water andapplied to a 7.2 × 60 cm. column packed with a microcrystallinecellulose product (Avicel, sold by FMC Corporation), suspended in 70percent aqueous acetonitrile, and washed with acetonitrile prior toaddition of the active sample. After application of the sample, thecolumn was washed with one column volume of acetonitrile, and theactivity was eluted with methanol. The active fractions were combinedand concentrated to approximately 200 milliliters, and the activity wasprecipitated by the addition of 10 volumes of acetone. The precipitatewas filtered, washed with acetone, and dried in vacuo. The yield was9-12 g.

Twenty grams of material obtained as described above was dissolved in aminimum of water and applied to a silica gel column (7.2 × 60 cm.). Thesilica gel (Grade 950 produced by Davison Chemical) was previouslywashed with water, then methanol, and suspended in 70 percentacetonitrile for packing the column. After application of the sample,the column was washed with one column volume of acetonitrile, and theactivity was eluted with 70 percent acetonitrile. The most activefractions were combined, concentrated to dryness in vacuo, and dissolvedin methanol, and the activity was precipitated with 10 volumes ofacetone. The precipitate was filtered, washed with acetone, and dried invacuo. The yield was 8 g. Less active fractions yielded an additional 6grams.

EXAMPLE 9 PURIFICATION OF A16884 MONOAMMONIUM SALT

One gram of a freeze-dried preparation prepared as described in Example8 was dissolved in four milliliters of water and applied to a 2 × 60 cm.column packed with 175 milliliters of silica gel Grade 950 in 80 percentaqueous acetonitrile. The column was eluted with acetonitrile:water(4:1). The elution was followed by assay and paper chromatography. As aresult of the elution, a plurality of fractions was obtained. Thefractions containing antibiotic A16884 as the monoammonium salt werecombined, concentrated to dryness, dissolved in a small volume ofdimethylsulfoxide, then in several milliliters of ethanol, and theactivity was precipitated with the addition of excess ether. Theprecipitate was centrifuged and dried in vacuo. The yield of antibioticA16884 monoammonium salt was 91 mg.

EXAMPLE 10 PREPARATION OF ANTIBIOTIC A16884 IN ACID FORM

Two hundred milligrams of the monoammonium salt of A16884 were dissolvedin 30 milliliters of water, and 6 milliliters of Dowex 50 × 12(H+) resin(sold by the Dow Chemical Co.) were added. The mixture was stirred forthirty minutes, filtered, the resin washed with water on the filter, andthe filtrates were combined. The combined filtrate had a pH of 2.7. Thefiltrate was concentrated in vacuo to about 1 milliliter, 4 millilitersof methanol were added, and the acid was precipitated by the addition of40 milliliters acetone. The precipitate was removed by centrifugationand dried in vacuo yielding 35 milligrams of antibiotic A16884 in theacid form. It exhibited pK'a's of 3.5, 5.2, and 10.3 when titrated in 66percent dimethylformamide at an initial pH of 4.5.

EXAMPLE 11 PREPARATION OF DISODIUM SALT OF A16884

One hundred and eighty milligrams of A16884 monoammonium salt weredissolved in about 2 milliliters water and the pH was adjusted to 10with 1N NaOH. The solution was concentrated in vacuo to a low volume, 4milliliters methanol were added, and the disodium salt were precipitatedwith the addition of 40 milliliters acetone. The salt was removed bycentrifugation and dried in vacuo. It exhibited pK'a's of 3.9, 5.2, and10.5 when titrated in 66 percent dimethylformamide at an initial pH of10.4; and when analyzed by atomic absorption analysis, it showed 6percent sodium.

EXAMPLE 12 PREPARATION OF ANTIBIOTIC A16884 HYDROCHLORIDE

A16884 Monoammonium salt (200 mg.) was dissolved in two milliliters ofwater and adjusted to pH 2.0 with 1N HCl. The reaction mixture was thendiluted with 5.0 milliliters of methanol and 50 milliliters of acetoneand added to precipitate the desired antibiotic A16884 hydrochloride. Itwas separated by centrifugation, washed with acetone, and dried invacuo. Analysis showed 5.74 percent chlorine and electrometric titrationin 66 percent dimethylformamide at an initial pH of 5.0 showedtitratable groups at 3.9, 5.2, and 10.4.

EXAMPLE 13 ISOLATION OF CRUDE ANTIBIOTIC A16884 AS THE MONOSODIUM SALT

Approximately 60 liters of broth, obtained as reported in Example 7,were filtered with the aid of Hyflo-Super-cel. The broth filtrate waspassed over a 9.6 × 150 cm. column packed with carbon (Pittsburgh Cal.12 × 40). The column was washed with water until colorless, and theabsorbed activity was removed by passing 50 percent aqueous acetone overthe column. The fractions containing the activity were combined,concentrated in vacuo to remove the acetone, and applied to a 5.9 × 104cm. column packed with IRA-68 (acetate cycle). The column was washedwith water until the effluent was clear and colorless, and the activitywas removed by washing with 0.1M sodium acetate. The active fractionswere combined, and passed over a 4.3 × 72 cm. column packed withPittsburgh Cal. (12 × 40) carbon. The column was washed with six columnvolumes of water, and the activity was eluted with 30 percent aqueousacetone. The active fractions were combined, concentrated in vacuo toremove the acetone, and freeze dried. Yield of 20-30 g. Analysis showed2.5 percent sodium.

Antibiotic A16884, as the monoammonium salt, was evaluated for thecontrol of plant-pathogenic bacterial organisms. In this evaluation,antibiotic A16884 monoammonium salt was formulated in an aqueous sprayformulation at a concentration of 400 parts thereof per million parts byweight of ultimate composition. Thirty-day-old tomato plants were usedin the evaluation, 2 plants/pot. Plants in one pot were treated with thesolution, described above, were allowed to air dry, and were theninoculated with a medium sustaining an active growth of Pseudomonassolanacearum. The plants in the other pot were sprayed with an aqueousspray solution identical with the treating solution described above butlacking the antibiotic, to serve as a control. The plants serving ascontrol were likewise subsequently inoculated. All plants were held for24 hours in a moist chamber, then removed and held for 7 days under goodagricultural conditions. At the end of this seven-day period all of theplants were observed to determine presence, and if present, degree, ofinfection. The plants treated with antibiotic A16884 monoammonium saltwere completely free of symptoms of disease caused by Pseudomonassolanacearum, whereas the control plants exhibited extensive symptomsattributable to Pseudomonas solanacearum.

We claim:
 1. The method for treating internal parasitic stomach andintestinal worms in a warm-blooded animal which comprises administeringto such an animal in need of said treatment an effective, anthelminticamount of an active agent which is a compound of the formula ##SPC2##ora pharmaceutically acceptable salt thereof.
 2. The method of claim 1wherein the active agent is the monoammonium salt.
 3. The method ofclaim 1 wherein the active agent is the hydrochloride salt.
 4. Themethod of claim 1 wherein the active agent is the sodium salt.
 5. Themethod for treating Ascaris lumbricoides var. suum in a warm-bloodedanimal which comprises administering to such an animal in need of saidtreatment an effective, anthelmintic amount of an active agent which isa compound of the formula ##SPC3##or a pharmaceutically acceptable saltthereof.